Respiratory impairment during Pneumocystis Pneumonia (PcP) is closely related to exuberant pulmonary inflammation in response to the organism. Anti-inflammatory corticosteroids (in addition to antibiotics) improves outcome during PcP, but are associated with further immune suppression and co-infection. Our recent studies demonstrate that Pneumocystis cell wall components including 2-glucans (PCBG) interact with alveolar macrophages and epithelial cells stimulating release of cytokines and chemokines that promote inflammatory cell recruitment in the lungs. We further demonstrated that host cell lactosylceramide mediates inflammatory activation in response to Pneumocystis and purified PCBG. Furthermore, glucosylceramide synthase (GCS) inhibitors, which reduce lactosylceramide, not only strongly suppress lung inflammation during PcP, but also strongly suppress the numbers of Pneumocystis organisms in treated mice. Our data further support that Pc itself possesses GCS synthetic molecules necessary for organism viability. Thus, GCS inhibitors represent a potential new class of anti-Pneumocystis agents with both beneficial immune modulating activity as well as direct suppressive effects on Pc. We currently hypothesize that Pneumocystis activates deleterious inflammatory signaling in epithelial cells and macrophages through lactosylceramide mediated MAPK signaling with subsequent cytokine/chemokine generation. We further postulate that glycosphingolipid synthesis in the organism itself occurs through a PCGCS-1 synthetase essential for Pneumocystis viability. These concepts will be addressed through three independent, interrelated, aims. Aim 1 will evaluate the mechanisms by which Pneumocystis and PCBG activate MAPK signaling in macrophages and epithelial cells, resulting in release of inflammatory cytokines and chemokines. In Aim 2, we will define the GCS synthetic machinery of Pneumocystis by characterizing GSC-1 synthetase, and its role in Pc viability. Finally, Aim 3 will evaluate specific GCS inhibitors as both prophylactic and therapeutic regimens for PcP, defining their impact on lung inflammation, gas exchange, and organism burdens. Better understanding the roles of lactosylceramide related signaling during PcP will better define mechanisms of lung injury during this infection. More over, this proposal also represents the potential to develop a new class of anti-pneumocystis agents with important effects both on the organism, as well as on organism-driven lung inflammation, during this important infection. PUBLIC HEALTH RELEVANCE. Pneumocystis pneumonia remains a major cause of illness and death in patients with impaired host defenses, and is therefore of significant relevance for public health particularly in patients with AIDS, malignancies, and following organ transplantation. Lung inflammation during Pneumocystis pneumonia strongly contributes to lung injury and death during this infection. The following studies encompass the development of a new means to suppress both lung inflammation as well as the Pneumocystis organisms themselves during pneumonia.